Elution chromatography



Jan. 18, 1966 M. J. E. GOLAY 3,230,167

ELUTION CHROMATOGRAPHY Original Filed June 5, 1959 INVENTOR. [@7661 J E 6' 010g United States Patent 3,230,167 ELUTION CHROMATOGRAPHY Marcel J. E. Golay, Rumson, N.J., assignor to The Perkin-Elmer Corporation, Norwalk, Conn., a corporation of New York Continuation of application Ser. No. 818,366, June 5, 1959. This application July 6, 1962, Ser. No. 207,920 Claims. (Cl. 21031) This application is a continuation of my copending application Serial Number 818,366 for Chromatographic Apparatus, filed June 5, 1959, now abandoned.

This invention relates to chromatographic separating columns and, more particularly, to packed chromatographic preparative columns. Further, the invention relates to elution development as applied to either liquidsolid, gasliquid, or gas-solid chromatography.

One of the disadvantages of packed chromatographic columns results from the presence of inhomogeneities due to variations in packing material and other factors. These difliculties are most apparent in preparative columns which have greater cross-sectional areas than nor mally found in analytical columns. The net result of such inhomogeneities is to increase the height equivalent to a theoretical plate (HETP or 11) of the column and thereby decrease its efliciency and its resolution capabilities. The term HETP is well known to workers in this field and is fully defined in Keulemans, Gas Chromatography, Reinhold Publishing Co., 1957, particularly at pages 98, 113, and 114.

It is, therefore, the primary object of the present invention to provide improved packed chromatographic columns. Other objects are to provide such columns having improved eficiency and resolution and to provide means for improving the separation capabilities of existing chromatographic columns.

The above objects are achieved by providing a method of separating a multi-components sample fluid into its components which comprises passing said sample fluid, together with an inert carrier, through a first section of chromatographic column capable of selectively retarding the flow of at least some of its components. The fluid is passed from the first chromatographic section through a substantially unobstructed mixing passage and the mole cules of the fluid are randomly distributed thereby to reduce the eifect of column inhomogeneities.

The invention disclosed herein will be more readily apparent from the following description and appended claims taken in conjunction with the figures of the attached drawing wherein:

FIG. 1 is a partially schematic illustration of a packed chromatographic column embodying the features of the present invention;

FIG. 2 is an exploded view of one embodiment of a mixing washer of the present invention; and

FIG. 3 is an exploded view of another embodiment of a mixing washer of the present invention.

The following description relates specifically to gas chromatography. However, it is to be understood that the invention is equally adapted to use with a liquid moving phase and, insofar as this invention is concerned, the same principles apply to each.

THEORY OF THE INVENTION Relatively large packed chromatographic columns very often contain some type of systematic variation. For example, while molecules of a particular type travel a distance x along one side of the column, identical molecules travel a distance (1+E)x on the other side. This result may be due to a number of factors such as, for example, variations in packing and the increased passage areas along the smooth walls of a packed column. Assume that such a systematic particle density ditterence exists throughout the column due to some fabricating bias. The effect of such difference is to cause an increase in the width of the curve of component concentration versus distance traveled along the column. The second moment (or spread) of the component concentration curve due to this elfect would increase quadratically with the distance traveled along the column were it not for the mixing which occurs due to lateral gas difiusion. A discussion of the concept of spread is contained in my paper, Theory and Practice of Gas-Liquid Partition Chromatography with Coated Capillaries, appearing in the book, Gas Chromatography, published 1958 by Academic Press Inc. Because of this elfect, it will be assumed that a given gas packet spreads linearly only for the distance traveled along the column in the time during which lateral diffusion causes the molecules to diituse across the diameter d of the column. The distance of such travel is of the order of d v/D where v is the mean gas velocity and D is the diffusion coeflicient of the sample molecules under consideration in the carrier gas. The diffusion coefficients of gases and liquids may be found in a number of standard texts for example, the American Institute of Physics Handbook, edited by Dwight E. Gray, McGraw-Hill, 1957. The HETP of the column due solely to the difference in linear travel may be expressed as:

The foregoing incremental HETP adds to the normal HETP, h, of the packed column so as to give a total HETP of:

where h is the HETP of a theoretical ideal (or homogeneous) column at optimum v.

It may be assumed that the third term of the righthand side of the foregoing equation, representing the incremental increase, is much larger than the second term due to the fact that Ed is much large than 11 By optimizing v and neglecting the second term of the righthand side of Equation 2, we obtain:

It will be evident that for effective mixing of the gas stream within the connecting passageway, the time of passage of the molecules within the tube should be suflicient to permit any molecule to ditiuse across it. This time is of the order of:

where d is the diameter of the connecting tube or passageway. Within the mixing tube the gas has a velocity of:

The minimum effective length of the mixing tube or passage will then be:

The minimum effective mixing tube length will be seen to be independent of the tube diameter al and equal to the length of column traversed by a gas "packet during the length of time required for lateral diffusion across the column.

Let U designate the volumetric second moment of a component packet flowing in a tube of cross-section S. Then:

where u designates the specific second moment of the packet, connected with the HETP through the relation:

The volumetric second moment has the property of being an invariant of the component packet when the gas stream follows paths of varying cross-sections and when pressure changes may be neglected. During its travel through the mixing tube, the component packet suffers an incremental second moment U equal to:

Substituting for v and L their values given by (6) and (7), and dividing by the square of the cross-section area of the column (neglecting the occupancy of the packing grains) times the length L between washers, we obtain the contribution k of the mixing tube to the effective HETP of the column:

When v is of the order of 4D/h and when h d and d d, the first term of the right hand side is negligible,

and we obtain for the overall effective HETP of the col- The above equation defines the minimum HETP obtainable using the mixing washers of the present invention.

Comparison of (15) with (2) indicates that the column with mixing washers will have a shorter HETP and provide better resolution than the plain column if:

It is mathematically convenient so to select d that the pneumatic resistance of the mixing tube is equal to that of a column portion between two mixing Washers, as this serves to simplify an otherwise algebraically complicated optimization problem. A characteristic granular dimension h should also be defined, the resistance of the column being the same as if it were composed of several capillaries of diameter h connected in parallel and having combined cross-sections equal to the cross-section of the column.

With this convenient assumption regarding h the stipulation that the column and mixing tube resistances be the same may be written:

Ed will be larger than h and the use of mixing washers will be beneficial if:

h Ed

Since h is known to be appreciably smaller than h (22) will be amply satisfied. It will be seen that mixing washers will be beneficial in large preparative columns in which systematic variations across the columns cause an appreciable HETP increase.

From the foregoing, formulae may be derived for the optimum values of L. L and d These values will be seen to be independent of D:

ILLUSTRATED EMBODIMENTS In FIG. 1 a chromatographic column 50 is shown partially cut away to disclose the mixing washers 54 interposed between sections of column packing material 52. Column 50 is supplied with a suitable carrier gas, such as He or N through conduit 11 and sampling valve 17. Valve 17 may be of any type suitable for injecting a sample into the carrier. For example, a valve of the type shown in US. Patent 2,757,541 issued August 7, 1956, to E. S. Watson et al. The elutant leaves the column by line 19 and may pass to a suitable detector or collecting system (not shown). Each of assemblies 54 comprises; three major elements 10, 12 and 16, which may correspond to similarly numbered elements of the embodiment of FIG. 2. Mixing washers 54 are so designed that an uneven fluid front moving along column 50 is relatively uniformly collected and passed into an elongated unpacked tube. Random dispersion of the gas molecules in their passage through this secondary tube redistributes the molecules into an even front. The downstream element of each washer then redistributes the molecules of the gas packet into the chromatographic column. The net result is a reduction of the inequalities which would be introduced into the fluid front by inhomogeneities in the chromatographic column if there were no mixing washers and the slanted fronts were thus permitted to become successively more slanted in their travel through the column.

Mixing washers suitable for use in the present invention are illustrated in FIGURES 2 and 3. FIG. 2 illustrates a washer which comprises a collecting funnel and similar dispersion funnel 12. The tunnel assemblies are designed to collect the gas stream uniformly across the column cross-section and to uniformly redistribute the fluid on the downstream side. The funnels are assembled back to back with their necks 14 connected by a coil tube 16 of suitable length and diameter. Screens 17 are provided at either end of the assembly to support the packing material and prevent its interference with fluid flow into the mixing tube. FIG. 3 illustrates a variant embodiment comprising essentially three suitable channeled disks. Collecting disk 20 is provided on its upper surface 22 with grooves 24 so dimensioned that their flow impedance is less than the flow impedance of the column height over which the sample front is spread. Yet they should not have cross-sections so large that the travel time of sample molecules from the points farthest removed from the center to the center exceeds the travel time of sample molecules across the column height over which the sample front is spread. The grooves overlap and channel to a common entryway 26. Entry 26 leads to a spiral channel 28 cut in the bottom of disk 28 whereby the gas is led outward to exit 30. Center body section 32 is sandwiched between collecting disk 20 and dispersion disk 44 and contains hole 34 for leading the gas into the outer portion of spiral channel 36 of dispersion disk 44. Disk 44 is identical to disk 20. The fluid is conducted in a reverse manner to that described with respect tothe collecting disk, being passed through exit 38 and channels 4G to return to the packed column. A screen section 42 is provided at each end of the washer assembly to support the column packing particles and prevent their entry into the channels.

Example As an example of the application of the present invention to gas chromatography, assume a column of 5 cm. diameter (d) packed with particles of diatomaceous earth or crushed brick, the particles being lightly impregnated with a relatively non-volatile liquid such as 2,5-hexanedione. Assume the particles to be of such average size as to impart to the column of characteristic dimension h (as defined above) of .02 cm. Such a column is useful for the separation of a variety of low-boiling hydrocarbon compounds, including such closely similar materials as normaland iso butane. At a given temperature and degree of particle impregnation, the physical separation between the points of maximum concentration of two components (such as nand i-butane) will be directly proportional to the length of column over which the components have passed. The width of a single component band (or the distance between the two points of the band at which the concentration of a single component equals half the maximum value) however, will be proportional to the square root of the product of the height equivalent to a theoretical plate (HETP) and the length traveled. The ratio of the separation of the concentration maxima of two similar compounds to the average of the zone widths of the pure compounds is a useful measure of the degree of separation or component purification achieved by the column. Clearly, any reduction in the height equivalent to a theoretical plate will permit a given degree of separation to be achieved with a shorter column, with attendant savings in such practical quantities as pressure drop, analysis time, and packing material.

The selection of helium as the carrier gas will result in a value of 0.4 cmP/sec. for D, the dittusion coefficient of butane in helium. Assume a systematic inhomogeneity across the column defined by E =.1.

From Equation 22:

Therefore, mixing Washers will be beneficial.

From Equation 21:

iimmim X-01X 5X.0 =(.03) g.31 cm. which is the minimum 12 at optimum flow velocity and column length.

From Equation 23, the optimum column length may be found:

The internal diameter for optimum conditions is defined by Equation 25:

From Equation 3 it will be seen that the minimum HETP obtainable without the washers of the present invention is:

It will be noted that, in calculating dimensions for optimum conditions, the term D is cancelled out. This indicates that optimum dimensions are independent of the fluids being separated. When compared with the .31 cm. obtained by use of Equation 21 above it will be seen that the washers of the present invention have provided an improvement of approximately 4.5 to 1. This means that the same degree of separation of the components can be achieved with about 47% of the total length of packed column required in the absence of the washers of the present invention, with the attendant benefits described above.

The present invention is not limited to gas chromatography, but is also applicable to liquid-liquid or liquidsolid column chromatography. In liquid systems, the diffusion coefficients are much smaller, typically 0.1 to 1.0 cm. /day in aqueous solutions. However, operation at or very near the optimum velocity leads to dimensional considerations which are, as in the case of gas chromatography, essentially independent of the diffusion coefficient.

It is to be understood that the above description is intended to be illustrative and not limiting. Many variations and modifications may be eifected in the method and apparatus of the invention while still remaining within the scope and spirit thereof.

I claim:

1. The method of separating a multicomponent fluid into its components which comprises passing a fluid carrier through a first section of chromatographic column; injecting into said carrier a controlled volume of said sample fluid; selectively retarding the flow of at least some of said components in their passage through said column; passing said carrier from said first column section into a substantially unobstructed elongated passage; realigning the molecules of said sample fluid in said passage whereby the front of each component is substantially perpendicular to the longitudinal axis of said column; passing said components and fluid carrier from said passage through a second chromatographic column section; and selectively retarding the flow of at least some of said components through said second section whereby 7 said components are selectively impeded and separated from each other.

2. The method of separating a multicomponent sample fluid into its components which comprises passing a fluid carrier through a first section of chromatographic column having comminuted packing material therein; injecting into said carrier a controlled volume of said sample fluid; selectively retarding the flow of at least some of said components in their passage through said column and packing material; passing said carrier and components from said first column section into a substantially unobstructed elongated passage having a cross sectional area less than that of said column; realigning the molecules of said sample fluid in said passage whereby the front of each component becomes substantially perpendicular to the longitudinal axis of said column; passing the components and fluid from said passage through a second chromatographic column section having comminuted packing material therein; selectively retarding the flow of at least some of said components through said second section whereby said components are selectively impeded and separated from each other.

3. Apparatus for separating a multicomponent fluid into its components which comprises a plurality of alternately connected chromatographic separating column sections having a length up to d v/D, where d is the internal diameter of said column section, v is the mean axial velocity of the fluid therethrough, and D is the diffusion coelficient of a component of said sample in said carrier, and a plurality of substantially unobstructed elongated passages having a length at least d v/D.

4. Apparatus for separating a multicomponent fluid into its components which comprises a plurality of alternately connected packed chromatographic separating column sections having a length L up to d v/D, and a plurality of substantially unobstructed elongated passages having a length of at least d v/D and an internal diameter of at least and up 'to d(4.9E) where d is the internal diameter of said column section;

D is the diffusion coeflicient of a sample component in said carrier;

E is the maximum difference in axial path length traveled by equivalent fluid particles in the column while traversing a mean axial distance L;

h is a characteristic granular dimension of the packing material of said column; and

v is the mean axial velocity of the fluid through said column.

5. Apparatus for separating a multicomponent fluid into its components which comprises a plurality of alternately connected packed chromatographic separating column section having a length L substantially equal to and a plurality of substantially unobstructed elongated passages each having a length substantially equal to and an internal diameter substantially equal to (4 /d h E) where d is the internal diameter of said column section;

B is the maximum difference in axial path length traveled by equivalent fluid particles in the column while traversing a mean axial distance L; and

h is a characteristic granular dimension of the packing material of said column.

6. Apparatus for separating a multicomponent sample fluid into its components by elution chromatography which comprises a plurality of series-connected chromatographic column means, each of said column means characterized by having a length L up to d v/D; means for passing a fluid carrier sequentially through the series combination of column means; means for injecting said sample fluid into said carrier prior to its entrance into said series combination; and a plurality of substantially unobstructed elongated passage means in alternating series fluid flow relationship with said column means, said passage means characterized by a length of at least d v/ D and an internal diameter up to d where E is the maximum difference in axial path length traveled by equivalent fluid particles in said column means while traversing a mean axial distance L, d is the internal diameter of said column means, v is the mean axial velocity of fluid therethrough, and D is the diffusion coeflicient of the fluid.

7. Apparatus for separating a multicomponent sample fluid into its components which comprises a plurality of packed chromatographic column means, each of said column means characterized by having a length L up to d v/D where d is the internal diameter of said column means, v is the mean axial velocity of the fluid therethrough, and D is the diffusion coeflicient of the fluid; a plurality of substantially unobstructed elongated passage means in alternating series fluid flow relationship with said column means, said passage means characterized by a length of at least d v/D and an internal diameter of at least where h is a characteristic granular dimension of the packing material of said column means, and up to d (4.9E) where E is the maximum difference in axial path length traveled by equivalent fluid particles in said column means while traversing a mean axial distance L; means for passing a fluid carrier through the series combination of column means and passage means, and means for injecting said sample fluid into said carrier.

8. Molecular distribution apparatus for packed chro matographic columns which comprises fluid collecting means defining first fluid flow conduit means; unobstructed passage defining means in series fluid flow relationship with said first conduit means; and fluid dispersion means defining second fluid flow conduit means in series fluid flow relationship with said mixing passage defining means.

9. The apparatus of claim 8 wherein said passage has a length of at least d v/D where d is the internal diameter of the chromatographic column, v is the velocity of the fluid through said column, and D is the diffusion coefficient of the fluid, and an internal diameter between at least and up to d(4.9E) where h is a characteristic granular dimension of the packing material of said column,

and D is the difiusion coeflicient of the fluid, and an internal diameter of at least and up to d(4.9E) where 11 is a characteristic granular dimension of the packing material of said column,

and E is the maximum unbalance of a fluid front traveling along the column a distance L; and second substantially disk-shaped body member means defining second fluid con- 1 0 ducting channel means connected to the other end of said tubular fluid flow means to conduct fluid therefrom and distribute said fluid to said chromatographic column.

References Cited by the Examiner Gas Chromatography, by Keulmans, copyright 1957 by Reinhold Publishing Co., New York, pages 12, 18-19, and 98 relied upon.

Technique of Organic Chemistry vol. X, Fundamentals of Chromatography, by Cassidy, Interscience Publishers Inc., New York, 1957, pages 214-219 relied upon.

MORRIS O. WOLK, Primary Examiner. 

1. THE METHOD OF SEPARATING A MULTICOMPONENT FLUID INTO ITS COMPONENTS WHICH COMPRISES PASSING A FLUID CARRIER THROUGH A FIRST SECTION OF CHROMATOGRAPHIC COLUMN; INJECTING INTO SAID CARRIER A CONTROLLED VOLUME OF SAID SAMPLE FLUID; SELECTIVELY RETARDING THE FLOW OF AT LEAST SOME OF SAID COMPONENTS IN THEIR PASSAGE THROUGH SAID COLUMN; PASSING SAID CARRIER FROM SAID FIRST COLUMN SECTION INTO A SUBSTANTIALLY UNOBSTRUCTED ELONGATED PASSAGE; REALIGNING THE MOLECULES OF SAID SAMPLE FLUID IN SAID PASSAGE WHEREBY THE FROMT OF EACH COMPONENT IS SUBSTANTIALLY PERPENDICULAR TO THE LONGITUDINAL AXIS OF SAID COLUMN; PASSING SAID COMPONENTS AND FLUID CARRIER FROM SAID PASSAGE THROUGH A SECOND CHROMATOGRAPHIC COLUMN SECTION; AND SELECTIVELY RETARDING THE FLOW OF AT LEAST SOME OF SAID COMPONENTS THROUGH SAID SECOND SECTION WHEREBY SAID EOMPONENTS ARE SELECTIVELY IMPEDED AND SEPARAED FROM EACH OTHER. 